Plant food allergens are a widespread group of plant proteins comprising cupin and prolamin superfamilies as well as proteinaceous molecules of the plant defense system. The prolamin superfamily includes several important types of allergens of legumes, tree nuts, cereals, fruits, and vegetables, and the cereal alpha-amylase and protease inhibitors. Cereal prolamine are major storage proteins of the cereal grain endosperm and are named glutenins and gliadins in wheat, secalins in rye, and hordeins in barley. The proteinaceous alpha-amylase inhibitors are non-gluten proteins. Based on structural similarity, proteinaceous alpha-amylase inhibitors with plant origin are usually classified in six families including lectin-like, knottin-like, CM-proteins. Kunitz-like, c-purothionin-like, and thaumatin-like (Richardson, 1990). CM (Chloroform-methanol)-proteins are a large protein family from cereal seeds containing 120 to 160 amino acid residues and five disulfide bonds. They show a typical double-headed alpha-amylase/trypsin domain. This feature makes it possible that they inhibit the activity of alpha-amylase and trypsin-like enzymes. The alpha-amylase inhibitor 0.19 is one of the most studied inhibitor of this family; it has a broad specificity, and inhibits alpha-amylases from insects, birds and mammals.
Furthermore, plant defensive proteins also comprise protease inhibitors. Most plant storage organs such as seeds and tubers contain 1-10% of their total proteins as protease inhibitors with different biochemical and structural properties inhibiting different types of proteases. Protein inhibitors are classified based on the type of enzyme they inhibit: serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, or metallocarboxy-protease inhibitors.
WO 2011/137322 recently disclosed that members of the non-gluten alpha-amylase/trypsin inhibitor family contained in wheat and related cereals are strong inducers of innate immune response in human intestine, thereby contributing to diseases like celiac disease. Moreover, alpha-amylase/trypsin inhibitors are also key contributing factors to diseases or conditions like gluten sensitivity, irritable bowel syndrome, inflammatory bowel disease, but also non-intestinal inflammation.
Celiac disease, also known as celiac sprue, gluten-sensitive enteropathy, or gluten intolerance is one of the most frequent food intolerances worldwide, with highest prevalence in Europe, North and South America, and Australia. Celiac disease is an inflammatory disease of the upper small intestine in genetically predisposed persons triggered by the ingestion of wheat, barley, rye and their cross-related varieties leading to a mal-absorption syndrome.
Gluten is a common dietary protein present in wheat, barley, rye and their cross-related varieties. Gluten is a complex mixture of glutamine- and proline-rich glutenin and prolamine molecules, which is thought to be the responsible factor for celiac disease induction in sensitive human individuals. Because of their unusual structure, with a high proline and glutamine content, the gluten proteins are partly resistant to intestinal enzymes, which lead to several non-degraded immunogenic peptides that can be sensed by the intestinal immune system. Ingestion of such proteins by sensitive individuals produces flattening of the normally luxurious, rug-like, epithelial lining of the small intestine known to be responsible for efficient and extensive terminal digestion of peptides and other nutrients. Clinical symptoms of Celiac Sprue include fatigue, chronic diarrhea, mal-absorption of nutrients, weight loss, abdominal distension, anemia, as well as a substantially enhanced risk for the development of osteoporosis and intestinal malignancies (lymphoma and carcinoma). The disease has an incidence of approximately 1 in 200 in European and North American populations.
The current essential treatment of gluten intolerance is a permanent strict withdrawal of gluten form the diet which is difficult to maintain. It is however important to define two categories of gluten intolerance in order to understand how the illness is affected by enzyme action in the gut. Celiac sprue is an autoimmune condition, a genetic inflammatory disorder of the small intestine. When gluten proteins break down during digestion, they fragment. These protein fragments are called peptides. In celiac sufferers, an inappropriate immune system response in the small intestine is initiated by one type of peptide, and the intestinal cells are damaged.
A second type of gluten intolerance results when the gut is injured by something other than celiac disease—the negative effect of a bacteria or yeast infection, for example, resulting in the loss of the intestinal enzymes which in turn leads to poor gluten digestion. While supplementing individuals with enzymes may be beneficial to celiac sufferers, they must remain on a strictly gluten free diet because of the possible strength of the autoimmune reaction when traces of gluten are left undigested.
Using specific enzymes as supplement can be effective in minimizing the need for a gluten-free diet for those individuals at risk of developing gluten intolerance, for gluten sensitive individuals, or wherein gluten intolerance is due to gut injury.
The use of exogenous proteolytic enzymes for gluten detoxification has been one of the most promising strategies for celiac disease management. Such enzymes have been used in both pretreatment of gluten containing flours, and as supplements. Prolyl-endopeptidases are known gluten-digesting enzymes which have been shown to digest gliadin peptides (WO 2002/45524 and WO 2002/46381).
WO 2011/137322 disclosed the use of antibodies against alpha-amylase CM 3 in order to treat celiac patients or food compositions, and considers the use of protease as an alternative. However, it is silent with regard to a specific enzyme treatment to efficiently hydrolyze alpha-amylase/trypsin inhibitors in the gastrointestinal tract or in pretreatment of foodstuff derived from wheat barley, rye and their cross-related varieties.
The indolins and purothionins comprise a group of sulphur-rich and basic low molecular weight proteins that are present in the endosperm of several Graminae. For example, the sequence identity of alpha-purothionins from wheat, rye and barley is more than 80%. Purothionin is a member of an allergy family designated by the World Health Organization-International Union of Immunological Societies as “Tri a 37” and might represent a diagnostic marker for an increased risk of wheat-induced anaphylaxis (Pahr et al., J Allergy Cin Immunol., 132 (4), pp 1000-1003).
It would be desirable to provide a safe, effective and cost competitive way to degrade alpha-amylase/trypsin inhibitors in the gastro intestinal tract as well as in food compositions comprising such inhibitors in order to treat celiac disease, enhance gastrointestinal comfort in celiac or non-celiac gluten sensitive individuals, or to delaying the onset of gastrointestinal discomfort in non-celiac gluten sensitive healthy individuals by reducing the exposure of the gut to plant allergens and more specifically to alpha-amylase/trypsin inhibitors such as CM 3 and 0.19. The use according to the present invention not only solves the problem of gluten sensitivity/allergy, but also allows stabilizing foam when used to degrade amylase trypsin inhibitors in beer.